Rice transcription factor OsWRKY37 positively regulates flowering time and grain fertility under copper deficiency.

Efficient uptake, translocation, and distribution of Cu to rice (Oryza sativa) spikelets is crucial for flowering and yield production. However, the regulatory factors involved in this process remain unidentified. In this study, we isolated a WRKY transcription factor gene induced by Cu deficiency, OsWRKY37, and characterized its regulatory role in Cu uptake and transport in rice. OsWRKY37 was highly expressed in rice roots, nodes, leaf vascular bundles, and anthers. Overexpression of OsWRKY37 promoted the uptake and root-to-shoot translocation of Cu in rice under -Cu condition but not under +Cu condition. While mutation of OsWRKY37 significantly decreased Cu concentrations in the stamen, the root-to-shoot translocation and distribution ratio in brown rice affected pollen development, delayed flowering time, decreased fertility, and reduced grain yield under -Cu condition. yeast one-hybrid, transient co-expression and EMSAs, together with in situ RT-PCR and RT-qPCR analysis, showed that OsWRKY37 could directly bind to the upstream promoter region of OsCOPT6 (copper transporter) and OsYSL16 (yellow stripe-like protein) and positively activate their expression levels. Analyses of oscopt6 mutants further validated its important role in Cu uptake in rice. Our study demonstrated that OsWRKY37 acts as a positive regulator involved in the uptake, root-to-shoot translocation, and distribution of Cu through activating the expression of OsCOPT6 and OsYSL16, which is important for pollen development, flowering, fertility, and grain yield in rice under Cu deficient conditions. Our results provide a genetic strategy for improving rice yield under Cu deficient condition.

Primer extension refractory PCR: an efficient and reliable genome walking method.

Genome walking, a molecular technique for obtaining unknown flanking genomic sequences from a known genomic sequence, has been broadly applied to determine transgenic sites, mine new genetic resources, and fill in chromosomal gaps. This technique has advanced genomics, genetics, and related disciplines. Here, an efficient and reliable genome walking technique, called primer extension refractory PCR (PER-PCR), is presented. PER-PCR uses a set of primary, secondary, and tertiary walking primers. The middle 15 nt of the primary walking primer overlaps with the 3' parts of the secondary and tertiary primers. The 5' parts of the three primers are heterologous to each other. The short overlap allows the walking primer to anneal to its predecessor only in a relaxed-stringency PCR cycle, resulting in a series of single-stranded DNAs; however, the heterologous 5' part prevents the creation of a perfect binding site for the walking primer. In the next stringent cycle, the target single strand can be extended into a double-stranded DNA molecule by the sequence-specific primer and thus can be exponentially amplified by the remaining stringent cycles. The nontarget single strand fails to be enriched due to the lack of a perfect binding site for any primer. PER-PCR was validated by extension into unknown flanking regions of the hyg gene in rice and the gadR gene in Levilactobacillus brevis CD0817. In summary, in this study, a new practical PER-PCR method was constructed as a potential alternative to existing genome walking methods.

Comprehensive suppression of single-molecule conductance using destructive σ-interference.

The tunnelling of electrons through molecules (and through any nanoscale insulating and dielectric material 1 ) shows exponential attenuation with increasing length 2 , a length dependence that is reflected in the ability of the electrons to carry an electrical current. It was recently demonstrated3-5 that coherent tunnelling through a molecular junction can also be suppressed by destructive quantum interference 6 , a mechanism that is not length-dependent. For the carbon-based molecules studied previously, cancelling all transmission channels would involve the suppression of contributions to the current from both the π-orbital and σ-orbital systems. Previous reports of destructive interference have demonstrated a decrease in transmission only through the π-channel. Here we report a saturated silicon-based molecule with a functionalized bicyclo[2.2.2]octasilane moiety that exhibits destructive quantum interference in its σ-system. Although molecular silicon typically forms conducting wires 7 , we use a combination of conductance measurements and ab initio calculations to show that destructive σ-interference, achieved here by locking the silicon-silicon bonds into eclipsed conformations within a bicyclic molecular framework, can yield extremely insulating molecules less than a nanometre in length. Our molecules also exhibit an unusually high thermopower (0.97 millivolts per kelvin), which is a further experimental signature of the suppression of all tunnelling paths by destructive interference: calculations indicate that the central bicyclo[2.2.2]octasilane unit is rendered less conductive than the empty space it occupies. The molecular design presented here provides a proof-of-concept for a quantum-interference-based approach to single-molecule insulators.

Bridging PCR: An Efficient and Reliable Scheme Implemented for Genome-Walking.

The efficacy of the available genome-walking methods is restricted by low specificity, high background, or composite operations. We herein conceived bridging PCR, an efficient genome-walking approach. Three primers with random sequences, inner walker primer (IWP), bridging primer (BP), and outer walker primer (OWP), are involved in bridging PCR. The BP is fabricated by splicing OWP to the 5'-end of IWP's 5'-part. A bridging PCR set is constituted by three rounds of amplification reactions, sequentially performed by IWP, BP plus OWP, and OWP, respectively pairing with three nested sequence-specific primers (SSP). A non-target product arising from IWP alone undergoes end-lengthening mediated by BP. This modified non-target product is a preferentially formed hairpin between the lengthened ends, instead of binding with shorter OWP. Meanwhile, a non-target product, triggered by SSP alone or SSP plus IWP, is removed by nested SSP. As a result, only the target DNA is accumulated. The efficacy of bridging PCR was validated by walking the gadA/R genes of Levilactobacillus brevis CD0817 and the hyg gene of rice.

Semi-Site-Specific Primer PCR: A Simple but Reliable Genome-Walking Tool.

Genome-walking has been frequently applied to molecular biology and related areas. Herein, a simple but reliable genome-walking technique, termed semi-site-specific primer PCR (3SP-PCR), is presented. The key to 3SP-PCR is the use of a semi-site-specific primer in secondary PCR that partially overlaps its corresponding primary site-specific primer. A 3SP-PCR set comprises two rounds of nested amplification reactions. In each round of reaction, any primer is allowed to partially anneal to the DNA template once only in the single relaxed-stringency cycle, creating a pool of single-stranded DNAs. The target single-stranded DNA can be converted into a double-stranded molecule directed by the site-specific primer, and thus can be exponentially amplified by the subsequent high-stringency cycles. The non-target one cannot be converted into a double-strand due to the lack of a perfect binding site to any primer, and thus fails to be amplified. We validated the 3SP-PCR method by using it to probe the unknown DNA regions of rice hygromycin genes and Levilactobacillus brevis CD0817 glutamic acid decarboxylase genes.

Contributions of Support Point Number to Mirror Assembly Thermal Sensitivity Control.

Due to the extreme environmental temperature variations, solutions that enable ultra-low thermal sensitivity in a mirror assembly are crucial for high-performance aerial optical imaging sensors (AOIS). Strategies such as the elimination of the coefficient of thermal expansion (CTE) mismatch and the employment of a flexure connection at the interface cannot be simply duplicated for the application involved, demanding specific design constraints. The contributions of support point number to the surface thermal sensitivity reduction and support stiffness improvement have been studied. A synthetic six-point support system that integrates equally spaced multiple ultra-low radial stiffness mirror flexure units and assembly external interface flexure units has been demonstrated on a 260 mm apertured annular mirror that involves significant CTE mismatch and demanding support stiffness constraint. The surface deformation RMS, due to the 35 °C temperature variation, is 16.7 nm.

Permethylation Introduces Destructive Quantum Interference in Saturated Silanes.

The single-molecule conductance of silanes is suppressed due to destructive quantum interference in conformations with cisoid dihedral angles along the molecular backbone. Yet, despite the structural similarity, σ-interference effects have not been observed in alkanes. Here we report that the methyl substituents used in silanes are a prerequisite for σ-interference in these systems. Through density functional theory calculations, we find that the destructive interference is not evident to the same extent in nonmethylated silanes. We find the same is true in alkanes as the transmission is significantly suppressed in permethylated cyclic and bicyclic alkanes. Using scanning tunneling microscope break-junction method we determine the single-molecule conductance of functionalized cyclohexane and bicyclo[2.2.2]octane that are found to be higher than that of equivalent permethylated silanes. Rather than the difference between carbon and silicon atoms in the molecular backbones, our calculations reveal that it is primarily the difference between hydrogen and methyl substituents that result in the different electron transport properties of nonmethylated alkanes and permethylated silanes. Chemical substituents play an important role in determining the single-molecule conductance of saturated molecules, and this must be considered when we improve and expand the chemical design of insulating organic molecules.

Neonatal cytochrome P450 CYP3A7: A comprehensive review of its role in development, disease, and xenobiotic metabolism.

The human cytochrome P450 CYP3A7, once thought to be an enzyme exclusive to fetal livers, has more recently been identified in neonates and developing infants as old as 24 months post-gestational age. CYP3A7 has been demonstrated to metabolize two endogenous compounds that are known to be important in the growth and development of the fetus and neonate, namely dehydroepiandrosterone sulfate (DHEA-S) and all-trans retinoic acid (atRA). In addition, it is also known to metabolize a variety of drugs and xenobiotics, albeit generally to a lesser extent relative to CYP3A4/5. CYP3A7 is an important component in the development and protection of the fetal liver and additionally plays a role in certain disease states, such as cancer and adrenal hyperplasia. Ultimately, a full understanding of the expression, regulation, and metabolic properties of CYP3A7 is needed to provide neonates with appropriate individualized pharmacotherapy. This article summarizes the current state of knowledge of CYP3A7, including its discovery, distribution, alleles, RNA splicing, expression and regulation, metabolic properties, substrates, and inhibitors.

Paraspinal Muscle Segmentation Based on Deep Neural Network.

The accurate segmentation of the paraspinal muscle in Magnetic Resonance (MR) images is a critical step in the automated analysis of lumbar diseases such as chronic low back pain, disc herniation and lumbar spinal stenosis. However, the automatic segmentation of multifidus and erector spinae has not yet been achieved due to three unusual challenges: (1) the muscle boundary is unclear; (2) the gray histogram distribution of the target overlaps with the background; (3) the intra- and inter-patient shape is variable. We propose to tackle the problem of the automatic segmentation of paravertebral muscles using a deformed U-net consisting of two main modules: the residual module and the feature pyramid attention (FPA) module. The residual module can directly return the gradient while preserving the details of the image to make the model easier to train. The FPA module fuses different scales of context information and provides useful salient features for high-level feature maps. In this paper, 120 cases were used for experiments, which were provided and labeled by the spine surgery department of Shengjing Hospital of China Medical University. The experimental results show that the model can achieve higher predictive capability. The dice coefficient of the multifidus is as high as 0.949, and the Hausdorff distance is 4.62 mm. The dice coefficient of the erector spinae is 0.913 and the Hausdorff distance is 7.89 mm. The work of this paper will contribute to the development of an automatic measurement system for paraspinal muscles, which is of great significance for the treatment of spinal diseases.

Large Variations in the Single-Molecule Conductance of Cyclic and Bicyclic Silanes.

Linear silanes are efficient molecular wires due to strong σ-conjugation in the transoid conformation; however, the structure-function relationship for the conformational dependence of the single-molecule conductance of silanes remains untested. Here we report the syntheses, electrical measurements, and theoretical characterization of four series of functionalized cyclic and bicyclic silanes including a cyclotetrasilane, a cyclopentasilane, a bicyclo[2.2.1]heptasilane, and a bicyclo[2.2.2]octasilane, which are all extended by linear silicon linkers of varying length. We find an unusual variation of the single-molecule conductance among the four series at each linker length. We determine the relative conductance of the (bi)cyclic silicon structures by using the common length dependence of the four series rather than comparing the conductance at a single length. In contrast with the cyclic π-conjugated molecules, the conductance of σ-conjugated (bi)cyclic silanes is dominated by a single path through the molecule and is controlled by the dihedral angles along this path. This strong sensitivity to molecular conformation dictates the single-molecule conductance of σ-conjugated silanes and allows for systematic control of the conductance through molecular design.

Silane and Germane Molecular Electronics.

This Account provides an overview of our recent efforts to uncover the fundamental charge transport properties of Si-Si and Ge-Ge single bonds and introduce useful functions into group 14 molecular wires. We utilize the tools of chemical synthesis and a scanning tunneling microscopy-based break-junction technique to study the mechanism of charge transport in these molecular systems. We evaluated the fundamental ability of silicon, germanium, and carbon molecular wires to transport charge by comparing conductances within families of well-defined structures, the members of which differ only in the number of Si (or Ge or C) atoms in the wire. For each family, this procedure yielded a length-dependent conductance decay parameter, ß. Comparison of the different ß values demonstrates that Si-Si and Ge-Ge σ bonds are more conductive than the analogous C-C σ bonds. These molecular trends mirror what is seen in the bulk. The conductance decay of Si and Ge-based wires is similar in magnitude to those from π-based molecular wires such as paraphenylenes However, the chemistry of the linkers that attach the molecular wires to the electrodes has a large influence on the resulting ß value. For example, Si- and Ge-based wires of many different lengths connected with a methyl-thiomethyl linker give ß values of 0.36-0.39 Å-1, whereas Si- and Ge-based wires connected with aryl-thiomethyl groups give drastically different ß values for short and long wires. This observation inspired us to study molecular wires that are composed of both π- and σ-orbitals. The sequence and composition of group 14 atoms in the σ chain modulates the electronic coupling between the π end-groups and dictates the molecular conductance. The conductance behavior originates from the coupling between the subunits, which can be understood by considering periodic trends such as bond length, polarizability, and bond polarity. We found that the same periodic trends determine the electric field-induced breakdown properties of individual Si-Si, Ge-Ge, Si-O, Si-C, and C-C bonds. Building from these studies, we have prepared a system that has two different, alternative conductance pathways. In this wire, we can intentionally break a labile, strained silicon-silicon bond and thereby shunt the current through the secondary conduction pathway. This type of in situ bond-rupture provides a new tool to study single molecule reactions that are induced by electric fields. Moreover, these studies provide guidance for designing dielectric materials as well as molecular devices that require stability under high voltage bias. The fundamental studies on the structure/function relationships of the molecular wires have guided the design of new functional systems based on the Si- and Ge-based wires. For example, we exploited the principle of strain-induced Lewis acidity from reaction chemistry to design a single molecule switch that can be controllably switched between two conductive states by varying the distance between the tip and substrate electrodes. We found that the strain intrinsic to the disilaacenaphthene scaffold also creates two state conductance switching. Finally, we demonstrate the first example of a stereoelectronic conductance switch, and we demonstrate that the switching relies crucially on the electronic delocalization in Si-Si and Ge-Ge wire backbones. These studies illustrate the untapped potential in using Si- and Ge-based wires to design and control charge transport at the nanoscale and to allow quantum mechanics to be used as a tool to design ultraminiaturized switches.

Substrate sustained release-based high efficacy biosynthesis of GABA by Lactobacillus brevis NCL912.

BACKGROUND: Gamma-aminobutyric acid (GABA) plays a significant role in the food and drug industries. Our previous study established an efficient fed-batch fermentation process for Lactobacillus brevis NCL912 production of GABA from monosodium L-glutamate; however, monosodium L-glutamate may not be an ideal substrate, as it can result in the rapid increase of pH due to decarboxylation. Thus, in this study, L-glutamic acid was proposed as a substrate. To evaluate its potential, key components of the fermentation medium affecting GABA synthesis were re-screened and re-optimized to enhance GABA production from L. brevis NCL912. RESULTS: The initial fermentation medium (pH 3.3) used for optimization was: 50 g/L glucose, 25 g/L yeast extract, 10 mg/L manganese sulfate (MnSO4·H2O), 2 g/L Tween-80, and 220 g/L L-glutamic acid. Glucose, a nitrogen source, magnesium, and Tween-80 had notable effects on GABA production from the L-glutamic acid-based process; other factors showed no or marginal effects. The optimized levels of the four key components in the fermentation medium were 25 g/L glucose, 25 g/L yeast extract FM408, 25 mg/L MnSO4·H2O, and 2 g/L Tween-80. A simple and efficient fermentation process for the bioconversion of GABA by L. brevis NCL912 was subsequently developed in a 10 L fermenter as follows: fermentation medium, 5 L; glutamic acid, 295 g/L; inoculum, 10% (v/v); incubation temperature, 32 °C; and agitation, 100 rpm. After 48 h of fermentation, the final GABA concentration increased up to 205.8 ± 8.0 g/L. CONCLUSIONS: L-Glutamic acid was superior to monosodium L-glutamate as a substrate in the bioproduction of GABA. Thus, a high efficacy bioprocess with 205 g/L GABA for L. brevis NCL912 was established. This strategy may provide an alternative for increasing the bioconversion of GABA.

Generalizing cell segmentation and quantification.

BACKGROUND: In recent years, the microscopy technology for imaging cells has developed greatly and rapidly. The accompanying requirements for automatic segmentation and quantification of the imaged cells are becoming more and more. After studied widely in both scientific research and industrial applications for many decades, cell segmentation has achieved great progress, especially in segmenting some specific types of cells, e.g. muscle cells. However, it lacks a framework to address the cell segmentation problems generally. On the contrary, different segmentation methods were proposed to address the different types of cells, which makes the research work divergent. In addition, most of the popular segmentation and quantification tools usually require a great part of manual work. RESULTS: To make the cell segmentation work more convergent, we propose a framework that is able to segment different kinds of cells automatically and robustly in this paper. This framework evolves the previously proposed method in segmenting the muscle cells and generalizes it to be suitable for segmenting and quantifying a variety of cell images by adding more union cases. Compared to the previous methods, the segmentation and quantification accuracy of the proposed framework is also improved by three novel procedures: (1) a simplified calibration method is proposed and added for the threshold selection process; (2) a noise blob filter is proposed to get rid of the noise blobs. (3) a boundary smoothing filter is proposed to reduce the false seeds produced by the iterative erosion. As it turned out, the quantification accuracy of the proposed framework increases from 93.4 to 96.8% compared to the previous method. In addition, the accuracy of the proposed framework is also better in quantifying the muscle cells than two available state-of-the-art methods. CONCLUSIONS: The proposed framework is able to automatically segment and quantify more types of cells than state-of-the-art methods.

Extreme Conductance Suppression in Molecular Siloxanes.

Single-molecule conductance studies have traditionally focused on creating highly conducting molecular wires. However, progress in nanoscale electronics demands insulators just as it needs conductors. Here we describe the single-molecule length-dependent conductance properties of the classic silicon dioxide insulator. We synthesize molecular wires consisting of Si-O repeat units and measure their conductance through the scanning tunneling microscope-based break-junction method. These molecules yield conductance lower than alkanes of the same length and the largest length-dependent conductance decay of any molecular systems measured to date. We calculate single-molecule junction transmission and the complex band structure of the infinite 1D material for siloxane, in comparison with silane and alkane, and show that the large conductance decay is intrinsic to the nature of the Si-O bond. This work highlights the potential for siloxanes to function as molecular insulators in electronics.

Silver Makes Better Electrical Contacts to Thiol-Terminated Silanes than Gold.

We report that the single-molecule junction conductance of thiol-terminated silanes with Ag electrodes are higher than the conductance of those formed with Au electrodes. These results are in contrast to the trends in the metal work function Φ(Ag)<Φ(Au). As such, a better alignment of the Au Fermi level to the molecular orbital of silane that mediates charge transport would be expected. This conductance trend is reversed when we replace the thiols with amines, highlighting the impact of metal-S covalent and metal-NH2 dative bonds in controlling the molecular conductance. Density functional theory calculations elucidate the crucial role of the chemical linkers in determining the level alignment when molecules are attached to different metal contacts. We also demonstrate that conductance of thiol-terminated silanes with Pt electrodes is lower than the ones formed with Au and Ag electrodes, again in contrast to the trends in the metal work-functions.

High-Conductance Pathways in Ring-Strained Disilanes by Way of Direct σ-Si-Si to Au Coordination.

A highly conducting electronic contact between a strained disilane and Au is demonstrated through scanning tunneling microscope-based single-molecule measurements. Conformationally locked cis diastereomers of bis(sulfide)-anchor-equipped 1,2-disilaacenaphthenes readily form high-conducting junctions in which the two sulfide anchors bind in a bipodal fashion to one gold electrode, providing enough stability for a stable electrical contact between the Si-Si σ bond and the other electrode.

Tuning Conductance in π-σ-π Single-Molecule Wires.

While the single-molecule conductance properties of π-conjugated and σ-conjugated systems have been well-studied, little is known regarding the conductance properties of mixed σ-π backbone wires and the factors that control their transport properties. Here we utilize a scanning tunneling microscope-based break-junction technique to study a series of molecular wires with π-σ-π backbone structures, where the π-moiety is an electrode-binding thioanisole ring and the σ-moiety is a triatomic α-ß-α chain composed of C, Si, or Ge atoms. We find that the sequence and composition of group 14 atoms in the α-ß-α chain dictates whether electronic communication between the aryl rings is enhanced or suppressed. Placing heavy atoms at the α-position decreases conductance, whereas placing them at the ß-position increases conductance: for example, the C-Ge-C sequence is over 20 times more conductive than the Ge-C-Ge sequence. Density functional theory calculations reveal that these conductance trends arise from periodic trends (i.e., atomic size, polarizability, and electronegativity) that differ from C to Si to Ge. The periodic trends that control molecular conductance here are the same ones that give rise to the α and ß silicon effects from physical organic chemistry. These findings outline a new molecular design concept for tuning conductance in single-molecule electrical devices.

Mechanism for Si-Si Bond Rupture in Single Molecule Junctions.

The stability of chemical bonds can be studied experimentally by rupturing single molecule junctions under applied voltage. Here, we compare voltage-induced bond rupture in two Si-Si backbones: one has no alternate conductive pathway whereas the other contains an additional naphthyl pathway in parallel to the Si-Si bond. We show that in contrast to the first system, the second can conduct through the naphthyl group when the Si-Si bond is ruptured using an applied voltage. We investigate this voltage induced Si-Si bond rupture by ab initio density functional theory calculations and molecular dynamics simulations that ultimately demonstrate that the excitation of molecular vibrational modes by tunneling electrons leads to homolytic Si-Si bond rupture.

[Ginsenoside Rh2-induced inhibition of histone deacetylase 6 promotes K562 cells autophagy and apoptosis in vivo].

To study the in vivo inhibition effect of ginsenoside Rh2 on humanleukemia cells, and explore its mechanism from autophagy and apoptosis aspects, human leukemia K562 cells allograft tumor models were applied, and after administration of ginsenosides Rh2 by gavage, the tumor diameter, volume and inhibitory rate were measured, and the anti-tumor activity of ginsenosides Rh2 was observed. The levels of HAT and HDAC in tumor tissues were detected by chemical colorimetry assay, and expressions of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5 and HDAC6 were detected by Western blotting assay. The expression levels of vital genes closely associated with autophagy and mRNA expressions of HDAC6 and Hsp90 were detected by Real time-PCR. HE staining was used to observe apoptosis, and immunohistochemistry was used to detect the protein expressions of HDAC6, Hsp90 and activated caspases 3. The results showed that ginsenoside Rh2 could inhibit the growth of k562 cells allograft tumor, with a tumor inhibition rate up to 53.10%. Ginsenoside Rh2 could significantly decrease HDAC activity and decrease the expressions of HDAC1, HDAC2 and HDAC6, and inhibit the expressions of HDAC6 and HSP90, increase the expressions of vital autophagy genes (beclin-1, LC3A and LC3B). Histopathological results showed that ginsenosides Rh2 could significantly increase the tumor apoptosis. Therefore, ginsenoside Rh2 had good anti-tumor effect in vivo, and the mechanism maybe associated with regulating autophagy and apoptosis through HDAC6 and Hsp90 pathways and inhibiting the in vivo proliferation of tumor cells.

Electric field breakdown in single molecule junctions.

Here we study the stability and rupture of molecular junctions under high voltage bias at the single molecule/single bond level using the scanning tunneling microscope-based break-junction technique. We synthesize carbon-, silicon-, and germanium-based molecular wires terminated by aurophilic linker groups and study how the molecular backbone and linker group affect the probability of voltage-induced junction rupture. First, we find that junctions formed with covalent S-Au bonds are robust under high voltage and their rupture does not demonstrate bias dependence within our bias range. In contrast, junctions formed through donor-acceptor bonds rupture more frequently, and their rupture probability demonstrates a strong bias dependence. Moreover, we find that the junction rupture probability increases significantly above ∼1 V in junctions formed from methylthiol-terminated disilanes and digermanes, indicating a voltage-induced rupture of individual Si-Si and Ge-Ge bonds. Finally, we compare the rupture probabilities of the thiol-terminated silane derivatives containing Si-Si, Si-C, and Si-O bonds and find that Si-C backbones have higher probabilities of sustaining the highest voltage. These results establish a new method for studying electric field breakdown phenomena at the single molecule level.